Many proteins existing in vivo are glycoproteins, proteins having oligosaccharide chains. Oligosaccharide chains in glycoproteins work in such a way that they maintain the three-dimensional structures of the proteins, regulate solubility, and impart protease resistance thereto. It is now becoming evident that the oligosaccharide chains in glycoproteins are involved in life phenomena such as fertilization or differentiation, signal transduction, canceration, intracellular protein transport, and regulation of biological activities. Thus, oligosaccharide chains bonded to proteins play an important role in various physiological functions. However, these oligosaccharide chains have diverse structures, and they are classified into various categories. Under the circumstances, it is therefore extremely difficult to identify which oligosaccharide chain structure is involved in a life phenomenon. Synthesis of glycoproteins or glycopeptides having an oligosaccharide chain with a single structure is also indispensable for elucidating such functions. At present, glycoproteins can be expressed by biological approaches using protein expression, although glycoproteins having an oligosaccharide chain with a uniform structure are difficult to obtain. Therefore, studies have been made in recent years on the precise chemical synthesis of glycopeptides or glycoproteins having an oligosaccharide chain with a single structure.
The present inventors have established a process for preparing a large amount of a biantennary complex-type oligosaccharide chain that can be used as a raw material from a chicken egg by combining enzymatic and chemical methods (Patent Document 1) and a process for synthesizing a sialylated glycopeptide by applying a solid-phase peptide synthesis method to a complex-type oligosaccharide chain (Patent Document 2). If glycopeptides can be polymerized, large glycoproteins having an oligosaccharide chain with a single structure will be synthesized.
At present, the most effective peptide polymerization method is probably the native chemical ligation method (Non-Patent Document 1), which involves coupling a peptide fragment having cysteine (Cys) as an N-terminal amino acid to a peptide having thioester at the C-terminus.
Peptide synthesis methods generally used are solid-phase synthesis methods, which involve immobilizing an N-terminal protected amino acid onto an insoluble resin support, removing the protecting group in the amino acid, and then sequentially elongating a peptide chain. Examples of a method for producing the peptide having thioester at the C-terminus include a method which involves performing thioesterification during peptide excision from a solid phase and a method which involves subjecting the C-terminal carboxyl group of a peptide to thioesterification after peptide excision from a solid phase.
For example, a method which involves producing a peptide using a safety catch linker on a solid-phase resin and allowing a thiol compound to act thereon (Non-Patent Documents 1 and 2) is known as a method for performing the thioesterification during peptide excision from a solid phase. However, this method has many problems such as poor condensation efficiency in the immobilization of a first amino acid onto a resin, the slight racemization of amino acids during the condensation, and the poor reactivity of the thiol compound in esterification. Moreover, when a hydroxyl group of an oligosaccharide chain in a glycopeptide is unprotected, alkylation performed for activating the safety catch linker also alkylates the sugar hydroxyl group easily. Thus, dealkylation treatment must be performed. This treatment may influence glycosylation and so on, depending on conditions, and a uniform oligosaccharide chain structure cannot be secured in the obtained glycopeptide. To solve this problem, it is suggested that the hydroxyl group of the oligosaccharide chain is protected in advance. However, this approach is not efficient due to additional protection and deprotection steps.
A strong acid such as 95% trifluoroacetic acid or hydrogen fluoride is usually used for excising a peptide from a solid-phase resin. However, the use of such a strong acid involves the deprotection of peptide side chains or the cleavage of an oligosaccharide chain linkage in glycopeptides. A method using a trityl resin as a solid phase and acetic acid for excision (Non-Patent Documents 3, 4, and 5) and a method using a 4-hydroxymethyl-3-methoxyphenoxybutyric acid-modified resin (HMPB resin) as a solid phase and 1% trifluoroacetic acid (TFA) for excision (Non-Patent Document 6) have been reported as methods for excising a peptide from a solid-phase resin using a weak acid without causing deprotection. However, the method using a trityl resin cannot produce glycopeptides having an unprotected hydroxyl group. On the other hand, when a glycopeptide is prepared using the HMPB resin as a solid phase, the use of 1% TFA cannot excise the glycopeptide. Alternatively, the use of 10% TFA also causes the partial removal of protecting groups in the peptide side chains. For peptide thioesterification, particularly, the protection of the thiol group of N-terminal cysteine is essential for preventing self-condensation. Thus, deprotection during excision leads to fatal outcomes. Accordingly, these methods are not sufficient for producing a peptide having a carboxyl group, which is used as a raw material in the production of a peptide having thioester at the C-terminus.
A thioester form of a peptide can be produced by reacting a peptide having protected side chains with alkylthiol. However, this approach has the problem of C-terminal amino acid racemization. For circumventing racemization, a method which involves replacing a C-terminal amino acid by glycine (Non-Patent Document 7), a method using benzotriazol-1-yloxy-trispyrrolidinophosphonium hexafluorophosphate (PyBOP)/diisopropylethylamine (DIPEA) as a condensing agent in dichloromethane (DCM) (Non-Patent Document 8), and a method using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/DIPEA as a condensing agent in tetrahydrofuran (THF) (Non-Patent Document 9) have been reported. However, the method which involves replacing a C-terminal amino acid by glycine has a natural limit to the types of peptides that can be produced. Moreover, glycopeptides having a hydroxyl group that is not protected with a protecting group cannot be dissolved in the solvent such as DCM or THF. Thus, these solvents must be changed, although the C-terminal amino acid racemization becomes a problem again.    [Patent Document 1] WO 03/008431    [Patent Document 2] WO 2004/005330    [Non-Patent Document 1] J. Am. Chem. Soc., 121, 11369-11374 (1999)    [Non-Patent Document 2] Angew. Chem. Int. Ed., 44, 1650-1654 (2005)    [Non-Patent Document 3] Tetrahedron Lett., 38, 6237-6240 (1997)    [Non-Patent Document 4] Tetrahedron Lett., 44, 3551-3554 (2003)    [Non-Patent Document 5] J. Am. Chem. Soc., 123, 3885-3891 (2001)    [Non-Patent Document 6] Tetrahedron, 49, 9307-9320 (1993)    [Non-Patent Document 7] Tetrahedron Lett., 38, 6237-6240 (1997)    [Non-Patent Document 8] Tetrahedron Lett., 44, 3551-3554 (2003)    [Non-Patent Document 9] J. Am. Chem. Soc., 123, 3885-3891 (2001)
An object of the present invention is to provide a process for producing a peptide having a carboxyl group at the C-terminus, with protecting groups in the peptide side chains maintained, which is applicable to a non-glycosylated peptide or even to a glycopeptide having an oligosaccharide chain, particularly, an oligosaccharide chain with an unprotected hydroxyl group.
Another object of the present invention is to provide a process for efficiently producing a peptide thioester compound, with racemization reduced, which is applicable to a non-glycosylated peptide or even to a glycopeptide having an oligosaccharide chain, particularly, an oligosaccharide chain with an unprotected hydroxyl group.